WO2007092434A2 - Utilisation de detecteur impulsionnel a effet resistif avec des pores submicrometriques ou nanometriques pour la detection d'ensemble d'objets submicrometriques ou nanometriques - Google Patents

Utilisation de detecteur impulsionnel a effet resistif avec des pores submicrometriques ou nanometriques pour la detection d'ensemble d'objets submicrometriques ou nanometriques Download PDF

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WO2007092434A2
WO2007092434A2 PCT/US2007/003133 US2007003133W WO2007092434A2 WO 2007092434 A2 WO2007092434 A2 WO 2007092434A2 US 2007003133 W US2007003133 W US 2007003133W WO 2007092434 A2 WO2007092434 A2 WO 2007092434A2
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submicrometer
complex
pore
detecting
solution
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PCT/US2007/003133
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WO2007092434A8 (fr
WO2007092434A3 (fr
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Michael Mayer
Jeffrey D. Uram
Kevin Ke
Alan J. Hunt
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The Regents Of The University Of Michigan
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Publication of WO2007092434A3 publication Critical patent/WO2007092434A3/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores

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  • the present disclosure relates to methods for detecting the solution-based assembly of complexes from submicrometer or nanometer sized objects, including the active assembly of complexes and/or preassembled complexes, using a submicrometer pore, submicrometer tube or channel, nanopore, nanotube or channel, and the resistive-pulse technique.
  • complexes of submicrometer and nanometer sized objects of interest may include self- assembling complexes and/or assembly of complexes comprising different objects. Complexes may also include coupling between monovalent objects or complexes of polyvalent objects, including several objects to even thousands of objects or more.
  • assembly of complexes of submicrometer or nanometer sized objects may include complexes formed by protein-protein interactions, protein-virus interactions, nanoparticle-protein interactions, nanoparticle-virus interactions, nanoparticle-nanoparticle interactions, nanoparticle-template interactions, binding of monoclonal or polyclonal antibodies to antigens, binding of monoclonal or polyclonal antibodies to antigens immobilized on objects, polynucleotide-polynucleotide interactions, and protein-polynucleotide interactions, to name a few.
  • SEM scanning electron microscopy
  • TEM transmission electron microscopy
  • AFM atomic force microscopy
  • light scattering techniques e.g. dynamic light scattering
  • Detection labels including radioisotopes, chemiluminescent conjugates, or colorimetric assays, may present handling issues, short lifespans, and may alter the structure and/or binding characteristics of the object of interest depending on the location and/or nature of the conjugation.
  • some of these methods use indirect measurements of complex assembly, for example, by using secondary antibodies and/or label or detection affinities independent of the binding event of the objects of interest.
  • a method that is non-destructive and does not require immobilization or modification of the object would be advantageous. As such, methods for examining submicrometer or nanometer sized complexes in their native state would further preserve material for reuse or further analysis.
  • the technique discussed here is non-destructive, does not require but can function with immobilization, examines each complex individually in solution, and can measure hundreds of complexes in a matter of minutes.
  • the present technology provides a method for detecting the assembly of complexes.
  • the method comprises providing a solution where a first portion is separated from a second portion via a single submicrometer pore, a single submicrometer channel or tube, a single nanopore, or a single nanochannel or tube.
  • a submicrometer pore can also be a submicrometer channel or submicrometer tube and a nanopore can also be a nanochannel or nanotube.
  • One or more submicrometer or nanometer sized object(s) is added to the first portion of the solution. Due to molecular interactions, these objects assemble to form complexes consisting of two or more submicrometer or nanometer sized objects.
  • a complex Passage of a complex from the first portion of the solution through the submicrometer pore or nanopore to the second portion of the solution is detected using resistive-pulse sensing.
  • complexes comprising different numbers of submicrometer or nanometer sized objects are detected.
  • a complex includes at least two submicrometer or nanometer sized objects, and in some embodiments, the at least two submicrometer or nanometer sized objects may be the same object or may be different objects.
  • the method uses resistive-pulse sensing to detect a change in current, wherein the change is proportional to the volume of the complex. In some embodiments, the method uses resistive-pulse sensing to detect a number of resistive-pulses per time interval, wherein the number is correlated to the concentration of the complex. And in some embodiments, the method uses resistive-pulse sensing to detect a residence time of the complex in the submicrometer pore or nanopore, wherein the residence time is correlated to the velocity of the complex. Some embodiments also include a method that uses resistive-pulse sensing to detect blockage of the submicrometer pore or nanopore by the complex. In some embodiments, the complex may comprise detecting formation of complexes in real-time and/or may comprise detecting preassembled complexes.
  • the present technology also includes a method for identifying intermolecular interactions.
  • the method includes partitioning an electrolyte volume with a submicrometer pore or nanopore and establishing a concentration gradient of a submicrometer object across the submicrometer pore. A change in electrical signal is measured when a complex comprising at least two submicrometer objects traverses the submicrometer pore.
  • Some embodiments include measuring a change in electrical signal when a complex comprising at least two submicrometer or nanometer sized objects traverses the submicrometer pore.
  • the measuring may further include determining the volume of the complex based on the change in current, determining the concentration of complex based on the number of resistive pulses per time interval, and/or determining the velocity of the complex based on the residence time of the complex in the submicrometer pore or nanopore.
  • aspects of the present technology include a method that uses a submicrometer pore or nanopore to detect and characterize immune complexes consisting of proteins, such as staphylococcal enterotoxin B (an agent with bioterrorism potential) and polyclonal antibodies.
  • Other aspects include methods for detecting and characterizing complexes assembled from submicrometer particles or nanoparticles.
  • Further aspects include methods that use a submicrometer pore-based resistive-pulse sensor to 1) detect a specific virus or a virus specific antibody in solution, 2) probe the ability of an antibody to immunoprecipitate the virus, 3) determine the number of antibodies bound to individual virus particles, and 4) monitor the assembly of nanoparticles onto templates (e.g., antibodies onto viruses) in situ.
  • Still further aspects include methods that use resistive-pulse sensing to estimate the affinity constant of a biological or synthetic interaction between two submicrometer or nanometer sized objects, for example such as for an antibody binding to its antigen.
  • Other aspects include estimating the number of one submicrometer or nanometer sized object bound to another submicrometer or nanometer sized object in a complex.
  • Further aspects include methods for detecting and determining the solubility of submicrometer or nanometer sized objects such as drug molecules or proteins and probing the crystallinity of the complexes that form.
  • the present technology affords several benefits including: the ability to detect the assembly of the complexes in real-time and/or to detect preassembled complexes; detection of complexes formed from objects in their native state; detection, characterization, and quantification of the complexes, such as an examination of the binding of antibodies to viruses; and the ability to estimate and/or determine the affinity constants of the interaction of two objects.
  • the present methods are rapid, label free, may require no immobilization or modification of the object of interest, and may achieve single- complex sensitivity by monitoring changes in electrical resistance when the complexes pass through the submicrometer pore or nanopore.
  • the complex of interest may be detected in complicated media such as serum.
  • submicrometer pore- or nanopore-based sensing of complexes may enable portable or high-throughput immunoassays for diagnostics and biodefense.
  • FIG. 1 illustrates laser-based fabrication of submicrometer pores with conical geometry
  • FIG. 2 illustrates a side a view of a resistive-pulse sensing setup according to one embodiment of the present teachings
  • FIG. 3 illustrates time courses of the formation of immune complexes in solution
  • FIG. 5 illustrates a time course of the current peak amplitudes and volumes of immune complexes
  • FIG. 6 illustrates a resistive-pulse sensing technique for detecting and characterizing the binding of antibodies to virus particles according to one embodiment of the present teachings
  • FIG. 7 illustrates detection of an antibody-virus interaction using a submicrometer pore
  • FIG. 8 illustrates kinetics of antibody binding at different rations of antibody to virus concentration and estimation of the maximum number of antibodies that can bind to the virus
  • FIG. 9 illustrates the number of monoclonal anti-streptavidin antibodies from mouse bound per streptavidin-functionalized colloid, r, vs. the initial concentration of antibody in solution;
  • FIG. 10 illustrates a plot of the number of antibodies bound per colloid at equilibrium, r, as a function of the free antibody concentration at equilibrium;
  • FIG. 11 illustrates nanopo re-based detection of protein aggregates and crystals according to one embodiment of the present teachings;
  • FIG. 12 illustrates SEM images of conical pores with diameters of 575 nm and 900 nm and determination of the relationship between peak amplitude and particle volume in conical pores with submicrometer diameter;
  • FIG. 13 illustrates histograms of the halfwidths of events caused by immune complexes and nanoparticles passing through submicrometer pores with conical geometry;
  • FIG. 14 illustrates power spectra of original current traces with and without events
  • FIG. 15 illustrates the effect of the cutoff frequency used for low-pass filtering on the peak amplitudes of current events during passage of immune complexes through a submicrometer pore
  • FIG. 16 illustrates microscope images to verify the specific formation of immune complexes
  • FIG. 17 illustrates blockage of the submicrometer pore with a diameter of 650 nm by large immune complexes
  • FIG. 18 illustrates time courses of the formation of immune complexes in a solution containing serum
  • FIG. 19 illustrates a schematic design of a conical pore and the recording setup according to one embodiment of the present teachings
  • FIG. 20 illustrates a plot of the average peak amplitude of the resistive-pulses caused by particles with a diameter of 100, 130, and 160 nm passing through a pore with a diameter of 575 nm versus particle volume;
  • FIG. 21 illustrates the determination of the bandwidth available during Coulter counting and the bandwidth required to resolve events
  • FIG. 22 illustrates the effect of the cutoff frequency used for low-pass filtering on the peak amplitudes of current events during passage of viruses through a submicrometer pore
  • FIG. 23 illustrates a close-up view of a single event due to the passage of a virus through the pore before and after decimation of data
  • FIG. 24 illustrates a histogram of the half-widths of events due to the passage of viruses at different bandwidths in the absence and presence of antiserum to demonstrate that the bandwidth and data decimation did not distort the recorded signals;
  • FIG. 25 illustrates a histogram of the peak amplitudes of 1395 events caused by PBCV-1 without antibody bound passing through the pore as shown in FIG. 19; and illustrates the frequency of events versus the concentration of virus;
  • FIG. 26 illustrates microscopic observation of antiserum, control serum, and of virus antibody complexes; and [0040] FIG. 27 illustrates a TEM image with individual measurements of the distance between virus particles in an aggregate.
  • the present technology may rapidly detect the assembly of complexes formed of submicrometer or nanometer sized objects, with or without immobilization or labeling of the object, by combining a submicrometer pore or nanopore with resistive-pulse sensing to monitor the formation of complexes in solution.
  • a "submicrometer pore,” as used herein, includes a submicrometer tube or channel, a nanopore, and nanotube or channel.
  • a "submicrometer object,” as used herein, includes a nanometer sized object.
  • Resistive-pulse sensing also known as Coulter counting, monitors the transient change in resistance (resistive-pulse) that occurs when a particle passes through a submicrometer pore filled with electrolyte.
  • Resistive-pulse sensing and “Coulter counting” are used synonymously, and may employ a Coulter counter device, which is also referred to simply as a Coulter counter.
  • Resistive-pulse sensing is used for detecting and analyzing microscale, and increasingly, nanoscale objects.
  • a Coulter counter increases with decreasing pore diameter and length, various techniques may be used for the fabrication of membranes that contain a single submicrometer pore or nanopore.
  • Pore-forming proteins in planar lipid bilayers (PLBs) may be used as nanopore sensors. Fabricated structures, in comparison, can offer a high degree of robustness and withstand environmental stress such as vibration, pressure, extreme pH, and elevated temperatures.
  • Fabricated nanopores and nanotubes may be used for resistive-pulse sensing to detect viruses, aggregation of colloids, DNA, nanoparticles, and proteins.
  • the present technology includes a nanomachining technique that employs femtosecond-pulsed lasers to fabricate submicrometer pore and nanopore structures in borosilicate glass coverslides. See FIG. 1 and Example 1 for an exemplary method of laser nanomachining. This technique has the advantage that it does not require masks, etching, or high vacuum and that it can fabricate in glass. Glass is an excellent substrate material owing to its low-noise properties, its chemical and mechanical robustness, and its amenability to surface functionalization.
  • laser nanomachining is able to fabricate complicated 3D structures in optically transparent substrates. This enables generation of pores with a conical geometry and diameters of 575, 650 (shown in FIG. 1b, c), and 900 nm. See Example 2 for exemplary scanning electron micrograph (SEM) images of the 575 and 900 nm pores.
  • the conical shape may produce low-resistance pores in thick (> 1 mm) membranes that have low electrical capacitance. Decreasing the resistance increases the amplitude of resistive pulses as well as the rate of transport through the pore for a given pore diameter. Lowering the capacitance can reduce electrical-current noise, which permits recording at high bandwidths and increases the sensitivity of the Coulter counter.
  • FIG. 1 An example of laser-based fabrication of submicrometer pores with conical geometry is shown in FIG. 1.
  • the panels show: a) femtosecond- pulsed lasers enabled nanomachining of conical pores in glass with diameters as small as 575 nm; b) scanning electron microscope (SEM) image looking into the 35-mm cylinder of a pore (see a); c) SEM image focused on the narrowest part of the pore (diameter: 650 nm).
  • SEM scanning electron microscope
  • the glass slide with the pores may be mounted onto a fluidic setup, as shown in FIG. 2, made of poly(dimethylsiloxane) (PDMS) to characterize their electrical properties and to perform affinity assays.
  • PDMS poly(dimethylsiloxane)
  • An exemplary composition for the recording buffer, resulting electrical resistances, and noise values of the pores are disclosed in Example 3.
  • the response of the nano-Coulter counter may be characterized by using synthetic nanoparticles.
  • the resistive pulse from a spherical particle is proportional to the volume of the particle (as long as the particle diameter is less than « 0.4 of the diameter of the pore).
  • objects or particles with diameters of 100, 130, and 160 nm are passed through a conical pore, a linear relationship is observed between the amplitude of the current peak and the particle volume, as disclosed in Example 2.
  • FIG. 2 shows a cross-section view of an embodiment of the experimental setup constructed in accordance with the present teachings.
  • a patch-clamp amplifier appJies a constant voltage and detects small changes in current (pA-range) with fast time resolution (10-50 kHz).
  • a poly(dimethylsiloxane) (PDMS) fluidic setup allows for replacement of solution on either side of the submicrometer pore.
  • Resistive pulses that occur when complexes pass through the pore are monitored to detect and characterize the complexes.
  • the exemplary antibody-antigen system investigated herein uses a goat anti-mouse antibody and a mouse monoclonal anti-baculovirus antibody as the antigen.
  • Example 5 discloses experiments confirming immune complexes by phase contrast and fluorescence microscopy.
  • Three different equimolar concentrations (15, 30, and 151 nm) of the antigen and the anti-mouse antibody were examined using a pore with a diameter of 650 nm.
  • the assay can detect immune complexes at a concentration of 151 nM and 30 nM as shown by the resistive pulses in FIG. 3b, c; no immune complexes at a concentration of 15 nM of antibody and antigen were detected.
  • FIG. 3b, c shows that the amplitudes of many resistive pulses, caused by the immune complexes formed at a concentration of 151 nM, were considerably larger than those formed at 30 nM. This result indicates that the immune complexes grew larger at 151 nM than they did at 30 nM, and may explain why no immune complexes could be detected at a concentration of 15 nM. Indeed, the immune complexes that formed at a concentration of 151 nM grew so large that they eventually blocked the pore.
  • the arrow indicates the onset of pore blockage; see also Example 6.
  • FIG. 3 shows time courses of the formation of immune complexes in solution.
  • the panels show: a) Control experiment with the antigen (mouse monoclonal anti-baculovirus antibody) and a nonspecific anti- rabbit antibody, both at a final concentration of 151 nM; b) At a final concentration of 151 nM of antigen and the specific anti-mouse antibody, detectable immune complexes formed rapidly and eventually blocked the pore (arrow). Note the y-scale of b) is ten times larger than the scale of the other traces owing to the large size of the immune complexes; c) At a lower antibody- antigen concentration (30 nM), detectable immune complexes formed but were smaller and did not block the pore.
  • Each current trace is composed of multiple, short-duration recordings (length 1- 2 s; see marked scale) that were taken from data files recorded during the course of the experiment; a small gap separates each of these short recordings.
  • the time in minutes, after the addition of anti- mouse or anti-rabbit antibody to the recording buffer that contained the antigen, is indicated above the beginning of each short recording.
  • These recordings therefore represent short "snapshots" of the current activity throughout the entire experiment of several minutes duration.
  • a pore with a diameter of 650 nm (FIG. 1 b, c) was used for these experiments.
  • a control experiment was performed by using the same antigen and a nonspecific goat anti-rabbit antibody at a concentration of 151 nM.
  • FIG. 4 shows the detection of staphylococcal enterotoxin B (SEB) by sensing the formation of immune complexes in media containing a complex sample matrix.
  • SEB staphylococcal enterotoxin B
  • each current trace is composed of multiple, short duration (length 2 s, see marked scale) recordings that were taken from data files recorded at different times during the experiment; a small gap separates each recording.
  • the time in minutes after the addition of anti-SEB serum, or SEB, to the recording buffer is indicated above the beginning of each short recording.
  • SEB staphylococcal enterotoxin B
  • sheep anti-SEB serum The addition of the anti- SEB serum to a solution containing SEB caused a large increase in the size and number of detectable aggregates (i.e., complexes) when compared with the anti- SEB serum alone, as shown in FIG. 4. Similar results were obtained with a second system that employed rabbit antiserum to detect a monoclonal antibody, as disclosed in Example 7. These results demonstrate that submicrometer pore- based sensors can detect immune complexes in media that contain complex samples such as blood serum.
  • submicrometer pore- or nanopore-based Coulter counting offers the possibility to evaluate specific properties of these complexes, such as their volume and growth rate. These properties are important as the size of an immune complex influences its physiological properties, for instance its clearance from circulation and its adherence to phagocytes. Studying polydisperse immune complexes is difficult owing to their large heterogeneity. Light-scattering techniques have been used; however, as they measure multiple particles at once, these techniques can be problematic for characterizing polydisperse samples. In contrast, Coulter counting measures each particle individually and therefore can provide information on the volume, polydispersity, and growth of the immune complexes with single-aggregate (i.e., single- complex) sensitivity.
  • single-aggregate i.e., single- complex
  • FIG. 5a, d shows that the increase in volume of immune complexes.
  • the general trend of the average peak amplitudes compares well with data obtained by light scattering.
  • the sigmoidal shape in FIG. 5d may be a consequence of a thermodynamically stable size of the immune complexes.
  • FIG. 5a, d shows that the standard deviation in. the amplitude of the current peaks increased significantly during the growth of the immune complexes, therefore indicating a strong increase in the polydispersity of the complexes.
  • FIG. 5b, e the majority of the immune complexes sensed shortly after the addition of antibody (FIG. 5b, e) had volumes that were comparable to complexes that were sensed after 8 min (FIG.
  • FIG. 5 shows the time course of the current peak amplitudes and volumes of immune complexes.
  • the panels show: a) Growth of immune complexes at a concentration of 151 nM of both antigen (mouse monoclonal anti-baculovirus antibody) and anti-mouse antibody. A first-order exponential function was fitted to the data.
  • the small letters in graph a) corresponds to the time points from which the histograms shown in b) and c) were extracted; b) Peak amplitudes and volumes recorded at 240 s after the addition of anti-mouse antibody; c) Peak amplitudes and volumes recorded 490 s after addition of anti- mouse antibody. Note that 74% of the complexes maintained their volumes compared to b); however, a small fraction of complexes reached volumes that were up to ten times larger than in b); d) Growth of immune complexes at a concentration of 30 nM. A sigmoidal function was fitted to the data.
  • the small letters in graph d) correspond to the time points from which the histograms shown in e) and f) were extracted, e) Peak amplitudes and volumes recorded at 610 s after the addition of anti-mouse antibody; f) Peak amplitudes and volumes recorded 2400 s after the addition of anti-mouse antibody. Note the occurrence of peak amplitudes with approximately two, three, and four times the change in current ( « 200, 300, 400 pA) of those shown in e). Each point in a), d) reflects the average amplitude and aggregate volume obtained from peaks over a period of 20 s.
  • the number of proteins in an aggregate may be estimated by assuming a molecular volume of 347 nm 3 for an immunoglobulin G antibody.
  • the volumes of the immune complexes sensed by the pore with a diameter of 650 nm and an antibody antigen concentration of 151 nM ranged from 2.1 x 10 5 to 6.0 x 10 6 nm 3 , which corresponds to aggregates of 610 to 17,300 proteins.
  • Submicrometer pore-based detection of immune complexes using the present technology offers a general, rapid, label-free, and solution- based method for the detection of any submicrometer object, protein, or particle that can be triggered to form a detectable assembly, while providing information on the volume, growth, and polydispersity of individual aggregates or complexes.
  • the detection limit of 30 nM for antigens compares favorably to other label-free detection techniques such as affinity capillary electrophoresis (ACE), gel-based immunoprecipitation, and direct immunoaggregation assays based on light scattering, all of which have detection limits between 10 and 1000 nM depending on the technique.
  • ACE affinity capillary electrophoresis
  • direct immunoaggregation assays based on light scattering
  • the technology presented herein may be particularly useful for in situ, quantitative monitoring of controlled assemblies of nanoparticles (e.g., monitoring the number of nanoparticles in a complex, the speed of the formation of the complexes, etc.), thereby addressing an urgent need in nanotechnology.
  • the present technology also includes the use of resistive-pulse sensing for the detection, characterization, and quantification of the binding of antibodies to intact virus particles.
  • the technology includes a nondestructive method for detecting virus-specific antibodies in solution and for determining the number of antibodies bound to an intact virus in a physiological buffer. This label-free technique is able to operate with virus concentrations as low as 5 x 10 7 particles/ml_ and establishes whether or not the antibody can aggregate (i.e., immunoprecipitate) the virus by forming a complex.
  • the approach uses laser-fabricated pores in glass and measures transient changes in current (so-called "resistive pulses”) by using Coulter counting experiments.
  • reaction volume was 40 ⁇ l_, but this value could be reduced to ⁇ 10 ⁇ L via the integration of microfluidics. Due to the small size of the pores, this approach could potentially be miniaturized and performed in parallel for high-throughput applications.
  • FIG. 1 In order to measure the resistive pulses caused by the passage of virus particles through the pore, a similar setup to FIG. 1 was used. It consisted of a patch-clamp amplifier with two Ag/AgCI electrodes and a conical pore with a diameter of 650 nm mounted in a poly-(dimethylsiloxane) (PDMS) fluidic setup The setup is disclosed in Example 8. The pore was fabricated in a borosilicate cover glass using a femtosecond-pulsed laser. Glass was chosen as the substrate because it is an excellent material for low-noise electrical recordings (i.e., low capacitance, low dielectric loss), and the conical shape of the pore provided enhanced sensitivity compared to cylindrical pores. Replacement of the solutions on either side of the pore was straightforward due to the fluidic setup, and the transparency of the entire assembly made it possible to observe the pore with a microscope when necessary.
  • PDMS poly-(dimethylsiloxane)
  • Example 9 Before examining the interaction of antibodies with virus particles, the response of the submicrometer pore to spherical nanoparticles of defined size and shape was characterized, as further disclosed in Example 9.
  • a spherical particle passing through a cylindrical nanopore creates a resistive pulse with a peak amplitude proportional to the volume of the particle (as long as the particle diameter was less than ⁇ 40% of the diameter of the pore).
  • the proportionality between current peak amplitude and particle volume for the conical pore was 3.9 x 10 "4 pA/nm 3 .
  • Virus particles are typically not perfectly spherical; however, experimental evidence suggests that the shape of particles that resemble spheroids does not influence the linear relationship between particle volume and peak amplitude. This linear correlation was used to estimate the change in the volume of PBCV-1 virus particles before and after antibody binding, shown in FIG. 6.
  • FIG. 6 illustrates the resistive-pulse technique for detecting and characterizing the binding of antibodies to virus particles.
  • the panels show: A) Detection of virus particles before addition of antibodies: Single virions passing through the laser-fabricated conical pore cause a transient reduction in current (resistive pulse) as shown by the spikes (events) in the current trace.
  • the dotted line represents the mean of a Gaussian curve fit to the distribution of the peak amplitudes of the events.
  • the concentration of the virus was 4 x 10 7 particles/mL and the average current passing through the pore for all experiments was « 140 nA.
  • B) Detection of virus particles after addition of antibodies Binding of antibodies to the virus increases the volume of the particle leading to an increase in the peak amplitude when the viruses pass through the pore.
  • the current trace displays events that were recorded 10-15 min after addition of the antiserum, which was at a final dilution of 0.001 x the original antiserum. If the antibody is capable of causing aggregation of viruses, this approach makes it possible to identify dimers (and larger complexes of virus particles) by detecting events with approximately twice (three times, etc.) the peak amplitude of individual viruses.
  • FIG. 7 shows the detection of an antibody-virus interaction using a submicrometer pore.
  • the panels show: A) Current versus time trace before addition of antiserum: The transient increases in resistance (events) that occurred when viruses passed through the pore led to transient reductions in current. The dotted line 71 represents the threshold used to distinguish events caused by the passage of viruses from current noise. B) Current versus time trace approximately 8 min after addition of antiserum: The mean peak amplitude was approximately 22% larger than the mean peak amplitude before addition of antiserum, whereas the four largest peaks were presumably due to aggregates of virus particles.
  • the inset represents data from control experiments; the histograms show events that occurred before (74) and 2.5-3.5 min (75), 7.5-8.5 min (76), and 13-15 min (77) after addition of serum from a rabbit that was not immunized (final virus concentration 4.4 x 10 8 particles/mL, final dilution of this control serum: 0.0013x the original control serum).
  • the inset histogram includes repeating data sets of reference numerals 74, 75, 76, and 77, in blocks of four, respectively. The change in the mean peak amplitude of the control experiments was ⁇ 6.5%.
  • FIG. 8 depicts the kinetics of antibody binding at different ratios of antibody to virus concentration and estimation of the maximum number of antibodies that can bind to the virus.
  • the final dilution of the antiserum or control serum was held constant at O.OOIx the original serum.
  • the panels show: A) Plot of the number of antibodies bound to virus particles versus time.
  • the final concentration of the virus was either 2.8 x 10 8 (squares) or 4.0 x 10 9 particles/mL (circles).
  • the triangles represent a control experiment with nonspecific rabbit serum and a virus concentration of 3.4 x 10 8 particles/mL.
  • the error bars reflect the error of the mean value from a Gaussian fit to a histogram of the peak amplitudes of at least 50 events.
  • the error bars were calculated by summing the standard deviation of the mean values of the Gaussian fits to histograms of the peak amplitudes.
  • FIG. 7C also shows a second peak in the histogram of the peak amplitudes upon addition of antibody to the virus particles.
  • the mean value of the Gaussian distribution fitted to the second peak was approximately twice that of the first peak. Since the antiserum that was used can cause aggregation of viruses (see also Example 13), the second peak may be caused by dimers of viruses that were linked by the divalent polyclonal IgG antibodies in the antiserum. Control experiments with serum from a rabbit that was not immunized caused only a small ( ⁇ 6.5%) change of the mean of a Gaussian fit to the peak amplitudes of the virus (FIG. 7C, inset), indicating that binding of nonspecific antibodies (or other proteins) to the viruses was minimal.
  • PBCV-1 is known to contain a major capsid protein which carries the primary epitope to which the polyclonal antiserum binds. PBCV-1 is enclosed in 5040 copies of this major capsid protein. Since the observed maximum number is close to the protein copy number, namely 4,200 ⁇ 450 antibodies bound to each virus particle, most of these primary epitopes were accessible for antibody binding.
  • the present technology may determine the number of antibodies bound to viruses in their native conformation.
  • the present method is label-free, nondestructive, requires no immobilization or modification of the virus or antibody, and can establish if the antibody is suitable for immunoprecipitation. Decreasing the diameter of the pore may allow the detection of virus-antibody interactions for viruses that have diameters less than 190 nm. Due to the specificity of most antibody-virus interactions, this method may be used to detect the presence of an antibody directed against a particular virus in complex media such as serum (here the anti-PBCV-1 antibody); it may therefore be useful for immunoassays and vaccine development.
  • the ability to determine the number of antibodies bound to a virus enables at least three important applications. First, it makes it possible to predict the efficacy of antibody-mediated neutralization of viruses. Second, the number of antibodies that are bound to a virus can be used for determining the antibody's affinity and the valency of binding. And third, antibodies binding to a virus particle represent an accessible example of a well- defined self-assembly; monitoring this assembly process may thus be useful as a model system for studying templated self-assembly. Such a system may promote other attempts of controlled nanoassemblies (e.g., fabrication of hierarchical nanostructures through the binding of nanoparticles to engineered templates).
  • controlled nanoassemblies e.g., fabrication of hierarchical nanostructures through the binding of nanoparticles to engineered templates.
  • aspects of present technology also include using resistive-pulse sensing to estimate i), the affinity constant between one object and another object, and ii) the solid phase affinity constant between one object and another object.
  • resistive-pulse sensing to estimate i), the affinity constant between one object and another object, and ii) the solid phase affinity constant between one object and another object.
  • a method described herein illustrates how the solid phase affinity constant of an antibody for its antigen immobilized on a particle may be determined using a micropore and the resistive-pulse technique. This method can be directly applied to resistive-pulse sensors that use submicrometer pores or nanopores.
  • Submicrometer pores and nanopores which may be used to detect individual proteins, also enable this method to be readily adapted to measuring the affinity constant of monovalent interactions including, but not limited to: Fab fragments binding to a virus particle, a monovalent ligand binding to a monovalent or polyvalent protein, a monoclonal antibody binding to a monovalent antigen, and nanoparticles (unmodified particles and particles modified with functional groups) binding to a synthetic or biological object (template).
  • pores also allow this method to be readily adapted to measuring the avidity constant of polyvalent interactions including but not limited to: polyclonal antibodies binding to antigens in solution, monoclonal antibodies binding to proteins in solution that have than one copy of the epitope, polyclonal or monoclonal antibodies binding to viruses, and nanoparticles binding to a synthetic or biological object (unmodified particles and particles modified with functional groups).
  • An immunoassay using a micropore and the resistive-pulse method may be used to detect the interaction of a monoclonal anti-streptavidin antibody with streptavidin-functionalized nanoparticles. Resistive-pulses can be recorded during the passage of these spherical colloids with a diameter of 510 nm through the pore. A constant concentration of colloids (1.2 x 10 9 particles mL ⁇ 1 ) can be incubated with an increasing concentration of the antibody, and the resistive-pulses from the colloids with bound antibody can be recorded and compared to the pulses before antibody binding. The diameter of the colloids at different concentrations of antibody can be estimated using the following equation since the pore had cylindrical geometry:
  • ⁇ l is the change in current from baseline
  • I is the baseline current flowing through the pore
  • D is the diameter of the pore
  • L is the length of the pore
  • d is the diameter of the colloid. Since the change in current from baseline increased with increasing concentration of antibody, the diameters of the nanoparticles appear to increase due to antibody binding. Assuming that the antibody-antigen interaction reached equilibrium (binding of polyclonal antibodies to virus particles typically reaches equilibrium within 13 min), it is possible to use resistive-pulse sensing to estimate here the solid phase affinity constants of e.g. antibody-antigen interactions.
  • Eq. (1) is based on spherical particles by proposing that the transient increase in the resistance of the pore is due to the displacement of a volume of conducting electrolyte by the spherical particle.
  • Eq. (1) it may be assumed that binding of antibodies to the colloids created a dielectric layer of antibodies which could be treated as an increase in the diameter of the colloids.
  • the thickness of this hypothetical confluent film (and therefore the mean increase in particle diameter) may depend on the extent of coverage of the colloids with antibodies.
  • FIG. 9 shows the number of monoclonal anti-streptavidin antibodies from mouse bound per streptavidin-functionalized colloid, r, vs. the initial concentration of antibody in solution.
  • the concentration of the colloids was held constant at 1.2x10 9 particles mL "1 .
  • This plot is based in part on analyzing and converting the data presented by Saleh, O.A., Sohn, L.L., 2003. Proc. Natl. Acad. Sci. U.S.A. 100, 820-824, which is incorporated herein by reference.
  • the dsDNA delivered a cloud of mobile counter ions into the pore due to its highly charged nature (two negative charges per base pair) which caused a transient decrease in resistance. Under certain conditions, these two effects are able to cancel each other out causing the passage of dsDNA through the pore to create no signal.
  • the effect due to the surface charge of the dsDNA depends on the length of the dsDNA and the length of the pore; if the length of the dsDNA is significantly shorter than the length of the pore, the effect of the surface charge on the amplitude of the resistive pulses is assumed to be negligible.
  • the length of the pore was 7-9 ⁇ m and the diameter of the colloids was 510 nm.
  • the valency of the antibody-antigen interaction must be considered. Given that the antigen was immobilized on the colloid, the antibody could bind in a monovalent or divalent fashion (i.e. one or two arms of the antibody could bind to streptavidin molecules). Both possibilities may be investigated as follows. [0082] In order to estimate the solid phase affinity constant, K a , of the antibody in the case of monovalent binding between the antibodies and the antigen at all concentrations of antibody, the binding equilibria of the antibody- antigen interaction studied by Saleh and Sohn was analyzed.
  • the colloids (with many antigen molecules covalently attached to their surface) may be considered analogous to macrornolecules that possess many identical binding sites for a single ligand.
  • the thermodynamics of such as system are straightforward. The derivation begins with the simplest case, the one in which the entire macromolecule possesses only a single binding site for the ligand. This situation is equivalent to the interaction of an antibody, Ab 1 with a colloid, C A g that would carry a single antigen (here streptavidin) molecule. This scenario can be described using the following equation:
  • the equilibrium for this reaction is characterized by K a , the equilibrium constant, which in this example represents the solid phase affinity constant of the antibody (in general, this equation represents the affinity of one object for another):
  • [C A gAb] represents the concentration of the complex between the antigen- functionalized colloid and the antibody at equilibrium
  • [C Ag ] represents the concentration of free colloids at equilibrium
  • [Ab] represents the concentration of free antibody at equilibrium.
  • the binding equilibria governed by Eq. (3) can be characterized by a binding isotherm of the form:
  • n the number of antigens immobilized on a colloid.
  • r and [Ab] are known or can be determined experimentally, Eq. (5) can be used to determine K a , and n.
  • the concentration of the colloids is typically known and was held constant at 1.2 x 10 9 particles rtiL "1 by Saleh and Sohn in all experiments (the concentration of the colloids may also be determined by the frequency of the resistive pulses).
  • the volume-based analysis of the Coulter counting data introduced in the present disclosure made it possible to calculate the number of antibodies bound per colloid at equilibrium, r, as shown in FIG. 9. Multiplying r by the concentration of colloids revealed the concentration of bound antibodies at equilibrium.
  • the concentration of free antibody at equilibrium, [Ab] was then obtained by subtracting the equilibrium concentration of bound antibodies from the initial antibody concentration.
  • FIG. 10 shows a graphical plot of the number of antibodies bound per colloid at equilibrium, r, as a function of the free antibody concentration at equilibrium, [Ab].
  • Scatchard plots, as shown in FIG. 10, are commonly used to assess the presence of divalent binding. If there was significant divalent binding, the Scatchard plot would be non-linear since at least two apparent solid phase affinity constants would determine the binding interaction.
  • the present technology can use resistive-pulse sensing to estimate the solid phase affinity constant for the binding of an antibody to a specific antigen or more generally the affinity constant for receptor- ligand interactions when the binding interaction is predominately monovalent (e.g. a monovalent ligand and a monovalent or polyvalent protein or receptor).
  • the system analyzed here is analogous to antibodies binding to intact virus particles or to the attachment of nanoparticles to templates (or other objects such as viruses).
  • the quantitative approach of the present disclosure makes it possible to estimate the solid phase affinity or the avidity constant of monoclonal antibodies or the affinity of Fab fragments for their binding to antigens on viruses (or other antigens that are intrinsically immobilized on nanoparticles or even floating in solution) in physiological conformation.
  • this method may be used for determining the number of nanoparticles attached to another object ( ⁇ .g a biological or synthetic template) and thus for extracting the average "association constant" of nanoparticle-template interactions. Obtaining these values for synthetic systems is difficult by established methods such as electron microscopy or atomic force microscopy. The present technology can therefore find use in characterization and fabrication of the next generation of functionally assembled nanodevices.
  • a submicrometer pore or nanopore-based assay may be used for detecting the formation of protein aggregates and crystals in solution.
  • Application of the present methods may monitor in real-time the formation of assemblies of protein and simple analysis of the data may determine whether or not protein crystals formed.
  • light scattering techniques are commonly used for studying various aspects of protein aggregation and crystal growth; however, due to the nature of this bulk measurement, it is not possible to determine a true distribution of protein assemblies from light scattering data.
  • resistive-pulse sensors detect individual particles and can therefore provide the true distribution of the protein assembles. This true distribution may be used for rapid determination of whether or not protein crystals formed under the given conditions. Due to the small footprint of nanopores and the low power, cost, and reagent requirements, the present methods and assays could be used in high- throughput or laboratory applications for drug discovery and protein research.
  • Methods include the following features.
  • the candidate solution for generating protein crystals is prepared and placed in a microliter-volume fluidic well (affording short mixing time and minimal reagent costs) that contains the nanopore. From this starting point, there are three possible outcomes: (i) the solution does not generate any protein aggregates or crystals; (ii) the solution generates protein aggregates which are detected by the nanopore and possess a specific distribution as shown in FIG. 11 A; and (iii) the solution generates protein aggregates and crystals which are detected by the nanopore; the resulting distribution is expected to be different from protein aggregates alone as shown in FIG. 11 B.
  • the assay proposed here allows integrating structure-based drug design based on protein crystallization with high-throughput screening techniques.
  • the combination of these two techniques provides a powerful drug search paradigm for pharmaceutical companies and increases their probability of finding leads for a target protein.
  • the present technology will yield the solubility of proteins. Solubility is an important parameter for the pharmaceutical industry and the exact same nanopore-based solubility assay can be applied to small molecule therapeutics.
  • Nanomachining using a femto-second pulsed laser A cover glass (Corning 0211 borosilicate, Fisher Scientific, Pittsburgh, PA) was fixed to a 3-axes microscope nanomanipulation stage (Mad City Labs, Inc., Madison, Wl). A few drops of water were placed on the upper side of the cover glass at the area that was to be machined (if the machining time was greater than 30 minutes, an aluminum compartment sealed with tape was used to minimize evaporation of water).
  • the laser a directly diode-pumped Nd:glass CPA laser system (Intralase Corp., Irvine, CA), was focused through the a 100x oil immersion microscope objective (N.A.
  • Pulses were used with a duration of 600-800 fs (femtoseconds) that were frequency doubled from 1053 nm to 527 nm.
  • the glass was machined by scanning the laser in circular patterns which removed material layer by layer. Since the subsequent layer was formed under water, machining always proceeded at the glasswater interface. The submicron pores were machined in a three stage process.
  • the following parameters were used for the pores: 35 ⁇ m cylinder machined with 60-80 nJ per pulse at a frequency of 1.5 kHz; wide part of the cone machined with 12-15 nJ per pulse at 1.5 kHz; tip of the cone machined with 8-13 nJ per pulse at 10 Hz.
  • the glass coverslides were left in water for 12 hours with the 35 ⁇ m cylinder facing down; this configuration facilitated settling of debris out of the pore.
  • the glass coverslides were cleaned in a fresh mixture of 3:1 concentrated sulfuric acid to 30% hydrogen peroxide for at least 15 minutes, prior to use.
  • Peak amplitude is proportional to the volume of spherical particles in submicron pores with conical geometry.
  • FIG. 12 shows SEM images of conical pores with diameters of 575 and 900 nm and determination of the relationship between peak amplitude and particle volume in conical pores with submicron diameter. The panels show: a) SEM image looking into the 35 ⁇ m cylinder of the pore with a diameter of 575 nm. The inset shows a close-up of the narrowest part of the pore, b) SEM image looking into the 35 ⁇ m cylinder of the pore with a diameter of 900 nm.
  • the inset shows a close-up of the narrowest part of the pore
  • the dotted line represents the mean current amplitude of the 9 peaks
  • TRIS Shelton Scientific, Shelton, CT
  • bovine serum albumin Sigma, St. Louis
  • Affinity-purified goat anti-mouse antibody (H+L) conjugate labeled with tetramethylrhodamine isothiocyanate (TRITC) and affinity-purified goat anti-rabbit antibody (H+L) conjugate labeled with TRITC (Zymed, San Francisco, CA) were diluted in recording buffer and filtered through either a 0.1 ⁇ m or 0.2 ⁇ m membrane filter.
  • the TRITC labels on these antibodies were not used for the submicron pore assays but were useful to perform control experiments of immunoprecipitation with fluorescence microscopy (see Figure S4).
  • the sheep anti-SEB serum and purified SEB (both from Toxin Technology, Sarasota, FL) were filtered through a 0.1 ⁇ m membrane filter.
  • the 100, 130, and 160 nm particles polystyrene microspheres functionalized with carboxyl groups, Bangs Laboratories, Fishers, IN) were used at a concentration of ⁇ 1x10 10 particles mL "1 in recording buffer.
  • a patch clamp amplifier was used (Axopatch 200B, voltage clamp mode, applied potential of either 0.2 V (FIG. 3, FIG. 18) or 0.15 V (FIG. 4), analog low-pass filter set to a 100 kHz cutoff frequency), a low noise digitizer (Digidata 1322, sampling frequency set to 500 kHz), and a computer with recording software (Clampex 9.2, all from Axon Instruments, Union City, CA) for data acquisition. [0098] Since the current was recorded at high bandwidth (-50 kHz), care was taken in the analysis of the data to avoid two possible problems: amplifier saturation, and recording digitized data with low signal-to-noise ratios (SNR ⁇ 1).
  • Amplifier saturation was avoided by ensuring that the currents including their high-bandwidth noise were at all times within the dynamic range of the amplifier and of the digitizer used.
  • the maximum recorded current with its RMS noise of ⁇ 0.06 nA was at all times below 180 nA; the dynamic range of the recording setup was ⁇ 200 nA.
  • the second problem, recording digitized data with low SNRs occurs if the amplitude of the signal of interest is considerably lower than the noise levels. Such low amplitude resolution can lead to inaccuracies during off-line analysis (e.g., event detection after filtering). This condition was, however, avoided since the lowest signal-to-noise ratio of the high bandwidth (-50 kHz) data recorded was 2:1 (peak amplitude: RMS noise).
  • peak amplitude of at least 5 times the RMS noise were included in the quantitative analysis of immune complexes (see below).
  • the noise recorded was most likely somewhat higher than the theoretical expectation due to other sources of noise such as amplifier noise and dielectric noise.
  • the experimentally recorded RMS noise value of 16 pA was low when considering the "large" current of 140 nA during the recordings in the present work. This low noise value confirms that the design of the pores and the material properties of the glass substrate are well-suited for Coulter counting of nanoscale objects.
  • Example 4 Determination of the time resolution required for accurate extraction of quantitative information from Coulter counting analysis. Extracting quantitative data from Coulter counting experiments requires careful design of the recording system and the pore since these two entities determine the bandwidth of the measurement. The bandwidth is one of the most important aspects of the recorded data because it determines the time resolution.
  • the time resolution of the measurement sets the upper bound of the "speed" at which changes in current can be recorded. That is, if a change in current occurs faster than the time resolution of the recording, then the recorded current "jumps" from one value to the next and the intervening information on how the current arrived at this value is lost.
  • the time resolution of the measurement determines the maximum resolution with which the resistive pulse of a particle can be observed while it passes though the pore. If it moves faster than the time resolution, then the peak amplitude of the resistive-pulse will be clipped. This clipping can cause inaccuracies in calculations that use the peak amplitude.
  • Another important aspect of recording data accurately is the sampling frequency. According to the Nyquist theorem, the sampling frequency should always be at least 4 times the bandwidth of the recording.
  • the maximum possible bandwidth that is obtainable in a Coulter counting experiment is determined by the access resistance of the pore and the capacitance of the substrate.
  • the resistance of the pore in parallel with the capacitance of the substrate that supports the pore forms a one-pole low-pass RC filter.
  • the maximum bandwidth of this filter can be estimated by the following equation:
  • FIG. 14 shows the power spectra of original current traces with and without events (here immune complexes). Both current traces were recorded at maximum bandwidth of the recording setup (-50 kHz). The power spectrum of the current trace with events 141 contained significantly more low frequency content than the power spectrum of the current trace without events 142. As determined from the plot, the maximum frequency component of the events was approximately 8 - 9 kHz. Therefore the current trace could be processed by low-pass filters with cutoff frequencies of 10 kHz (dotted line) without causing significant signal distortion. The average peak amplitude of the events in the current trace was 123 ⁇ 40 pA. The current traces were obtained from a pore with a diameter of 650 nm.
  • FIG. 15 shows the effect of the cutoff frequency used for low-pass filtering on the peak amplitudes of current events during passage of immune complexes through a submicron pore.
  • the panels show: a) Current trace with three events after filtering with a digital (Gaussian) low-pass filter with a cutoff frequency of 50 kHz. b) Same current trace after filtering with a cutoff frequency of 20 kHz. The difference in the peak amplitude of the events between trace a) and b) was due to the reduction in current noise (from 26 to 17 pA RMS) and not due to clipping of the peak as a result of the reduced filter cutoff frequency, c) Same current trace after filtering with a cutoff frequency of 10 kHz.
  • FIGS. 16d, e show microscope images to verify the specific formation of immune complexes.
  • the panels show: a) Control experiment with the monoclonal antibody from mouse against baculovirus (antigen) at a concentration of 1.33 ⁇ M. No protein aggregates were seen on the slide by phase contrast microscopy, b) Control experiment with the anti-mouse antibody from goat that was labeled with tetramethylrhodamine isothiocyanate (TRITC) at a concentration of 1.33 ⁇ M. No protein aggregates were seen on the slide by phase contrast microscopy, c) False colored fluorescence image of the same field of view as in b).
  • TRITC tetramethylrhodamine isothiocyanate
  • phase contrast image shows at least eight micron-sized immune complexes (indicated with white arrows), e) False colored fluorescence image of the same field of view as in d).
  • a typical fluorescent filter set for rhodamine, an exposure time of 1 s, and the maximum intensity of excitation of the lamp was used to capture this image. All of the images were captured with a CCD camera (Photometries CoolSnap HQ, Roper Scientific, Trenton, NJ) and processed using image analysis software (Metamorph, Universal Imaging, Downington, PA).
  • Blockage of submicron pores by biospecific formation of large immune complexes As shown in FIG. 17, blockage of the submicron pore, with a diameter of 650 nm, by large immune complexes was detected. At a concentration of 151 nM monoclonal antibody from mouse against baculovirus (here used as the antigen) and 151 nM anti-mouse antibody, the resulting immune complexes grew large enough that they clogged the pore as indicated by step-wise increases in electrical resistance (blockage started approximately 15 minutes after addition of anti-mouse antibody). This "immunospecific blockage" may be useful for simple detection of antibody-antigen interactions.
  • the graph is composed of several concatenated data files; a small gap separates each file. This method can of course be applied to nanopores as well.
  • FIG. 18 shows time courses of the formation of immune complexes in a solution containing serum.
  • the panels show: a) Control experiment with 2 ⁇ l_ serum from a rabbit that was not immunized (filtered through a membrane with pores of 0.1 ⁇ m) added to 40 ⁇ l_ of recording buffer. Note the presence of small peaks that were caused by serum components not removed by the filter.
  • Antigen here mouse monoclonal antibody against baculovirus
  • Anti-mouse antibody was dissolved in unfiltered rabbit serum and then this mixture was filtered using a membrane filter with 0.1 ⁇ m pores. A volume of 2 ⁇ l_ of rabbit serum containing anti-mouse antibody was added to 40 ⁇ l_ of recording buffer; the final concentration of antibody was 151 nM. Addition of the antigen to a final concentration of 355 nM caused a significant increase in the number of events and the size of the events, c) Antigen was added to a final concentration of 151 nM. As expected from FIG.
  • FIG. 19 shows a schematic design of the conical pore and the recording setup.
  • PDMS poly(dimethylsiloxane)
  • Recording buffer composed of 150 mM KCI, 50 mM TRIS buffer, pH 7.8, 0.1 mg mL ⁇ 1 bovine serum albumin, 0.1 % w/v Tween 20, was filtered through sterile, low-protein-absorption polyethersulfone membrane filters with a pore size of 0.2 ⁇ m (Pall, East Hills, NY). Concentrated PBCV-1 virions and the polyclonal antiserum from rabbit were both kindly provided by J. L. Van Etten (University of Kansas-Lincoln). The virus and antiserum were diluted in recording buffer and the antiserum solution was filtered through a 0.2- ⁇ m membrane filter.
  • Ag/AgCI pellet electrodes (Eastern Scientific, Rockville, MD) were used since the recorded currents were relatively large (» 140 nA).
  • a patch- clamp amplifier (Axopatch 200B) was used in voltage clamp mode and the analog low-pass filter was set to a cutoff frequency of 100 kHz.
  • the setup was completed by a low-noise digitizer (Digidata 1322, sampling frequency set to 500 kHz), and a computer with recording software (Clampex 9.2) for data acquisition.
  • Clampfit 9.2 (all from Axon Instruments, Union City, CA) was used.
  • Data was filtered with a digital Gaussian low-pass filter with a cutoff frequency of 10 kHz and then decimated to a sampling frequency of 50 kHz (see Example 10 for detailed analysis of the bandwidth of the measurement, the bandwidth and sampling frequency required to resolve an event due to a virus completely, and the effects of digital filtering and decimation of data on the peak amplitudes and half-widths of the events).
  • a peak was defined as an "event” due to passage of a virus if the signal had an amplitude of at least 13 times the standard deviation of the baseline signal from its mean for a duration of at least 25 ⁇ s and a maximum of 10 ms (all events had a halfwidth >100 ⁇ s).
  • the collected data was analyzed using Origin 7.5 (OriginLab, Northampton, MA) and Matlab (The MathWorks, Natick, MA).
  • FIG. 20 graphically depicts a plot of the average peak amplitude of the resistive-pulses caused by particles with a diameter of 100, 130, and 160 nm passing through a pore with a diameter of 575 nm versus particle volume.
  • the data were fitted using a linear regression algorithm that required the line to pass through the origin; the slope of the line was 4.2x10 '4 pA nm "3 .
  • a slope of 3.9x10 4 pA nm '3 was obtained for the pore with a diameter of 650 nm (FIGS. 19A, B).
  • Example 10 Determination of the bandwidth required for accurate extraction of quantitative information from Coulter counting analysis. Extracting quantitative data from Coulter counting experiments requires careful design of the recording system and the pore since these two entities determine the bandwidth of the measurement.
  • the bandwidth is one of the most important aspects of the recorded data because it determines the time resolution.
  • the time resolution of the measurement sets the upper bound of the "speed" at which changes in current can be recorded. That is, if a change in current occurs faster than the time resolution of the recording, then the recorded current "jumps" from one value to the next and the intervening information on how the current arrived at this value is lost.
  • the time resolution of the measurement determines the maximum resolution with which the resistive pulse of a particle can be observed while it passes though the pore. If the particle moves faster than the time resolution, then the peak amplitude of the resistive- pulse will be clipped. This clipping can cause inaccuracies in calculations that are based on the peak amplitude.
  • Another important aspect of recording data accurately is the sampling frequency. According to the Nyquist theorem, the minimum sampling frequency required to prevent aliasing is twice the signal bandwidth (i.e., if the signal has a bandwidth of 10 kHz, the sampling frequency must be at least 20 kHz); however, it is typically recommended that a sampling rate at least 5 times the signal bandwidth be used.
  • the maximum possible bandwidth that is obtainable in a Coulter counting experiment is determined by the geometry of the pore, the substrate material, the conductivity of the buffer, and the recording electronics.
  • the power spectrum of a high bandwidth current trace was examined.
  • the power spectrum contained a linear decrease in power between 4-1000 Hz and a roll-off in power after ⁇ 50 kHz.
  • the linear drop in the range of 4-1000 Hz is most likely due to Mf noise.
  • FIG. 21 illustrates the determination of the bandwidth available during Coulter counting experiments and the bandwidth required to resolve events.
  • the panels show: (A) Power spectra of current traces under three conditions: (211) - no digital filtering and an applied voltage of 0.2 V, (212) - no digital filtering and an applied voltage of 0 V, and (213) - same current trace used as in plot 211 after digitally filtering with a 1-pole RC filter with a cutoff frequency of 10 kHz. Based on these power spectra, the bandwidth of the Coulter counting apparatus (patch clamp amplifier and submicrometer pore) was -50 kHz.
  • the parameter that is important here namely the reduction of bandwidth due to the recording setup, can be obtained from the "roll-off' at higher frequencies.
  • the analysis of the power spectra in FIG. 22 therefore shows that the available bandwidth was ⁇ 50 kHz.
  • the pore can be modeled as a network of resistive and capacitive components, it is possible that the pore itself could act as a filter. Due to the geometry of the pore, the model circuit is complicated, and this result makes a direct derivation of the filtering characteristics (i.e., the transfer function) difficult. If the pore would constitute a significant filter, then it can be expected that the pore would act as a single pole (or multi pole) RC filter. In order to illustrate the hypothetical effect of such a filter, the original current trace was filtered with a single pole RC filter with a cutoff frequency of 10 kHz (arbitrarily chosen) and the power spectrum was recalculated, which is shown in FIG. 21 A as trace 213.
  • the current traces that contained events had more power in frequencies ranging from 4-8000 Hz compared to the trace that did not contain events. Therefore, a bandwidth of ⁇ 8 kHz was required to resolve the events completely. Due to this result, the RMS noise of the current traces was reduced by filtering with a digital low-pass filter (Gaussian) with a cutoff frequency of 10 kHz without causing significant distortion of events: as expected, the amplitude of the virus peaks was not significantly reduced ( ⁇ 5% decrease) by the 10 kHz filter when compared to the peaks that were filtered at 50 kHz as illustrated in FIGS.
  • a digital low-pass filter Gaussian
  • FIG. 22E demonstrates that filtering with a cutoff frequency as low as 5 kHz would have only reduced the peak amplitude of the signal by less than 11%. Significant reduction in amplitude would have been observed, however, if a cutoff frequency of 1 kHz was used as shown in FIG. 22F ( ⁇ 50% decrease).
  • the recorded data were filtered with a 10 kHz low-pass filter, and decimated to a sampling frequency of 50 kHz.
  • this decimation had a minimal effect on the peak amplitude (FIGS. 22C, D and FIG. 23 show that the decimation of data caused a negligible change in the peak amplitude of an event; the mean peak amplitude and the mean value of a Gaussian fit of over 200 events decreased by less than 1%).
  • Decimation also had a negligible effect on the event half-width (FIG. 23 and FIGS. 24B, C show that the decimation of data caused the mean half-width of over 200 events to decrease by less than 1 %).
  • FIG. 22 shows the effect of the cutoff frequency used for low-pass filtering on the peak amplitudes of current events during passage of viruses through a submicron pore.
  • the panels show: (A) Current trace with two events after filtering with a digital (Gaussian) low-pass filter with a cutoff frequency of 50 kHz. (B) Same current trace after filtering with a cutoff frequency of 20 kHz. (C) Same current trace after filtering with a cutoff frequency of 10 kHz. (D) Same current trace as in C but decimated to a sampling frequency of 50 kHz, instead of 500 kHz as in A-C. As predicted by the Nyquist sampling theorem, the amplitude of the signal did not change significantly (see FIG.
  • FIG. 23 illustrates a close-up view of a single event due to the passage of a virus through the pore before and after decimation of data.
  • the panels show: (A) Close-up view of a single event after filtering with a digital low- pass filter with a cutoff frequency of 10 kHz (sampling frequency of 500 kHz). (B) Same trace as in A decimated by a factor of ten (sampling frequency of 50 kHz). The change between the peak amplitude and half-width of trace A and trace B was smaller than 1%.
  • FIG. 24 graphically depicts histograms of the half-widths of events due to the passage of viruses at different bandwidths in the absence and presence of antiserum.
  • the graphs demonstrate that the bandwidth and data decimation used did not distort the recorded signals (i.e., was sufficient to resolve the entire signal).
  • the panels show: (A) Half-widths of events due to the passage of viruses after filtering with a digital Gaussian low-pass filter with a cutoff frequency of 50 kHz. (B) Same events as in A but filtered with a low-pass filter with a cutoff frequency of 10 kHz. (C) Same events as in B but after decimation to a sampling frequency of 50 kHz.
  • Example 11 [0132] Analysis of the measured diameter of PBCV-1 and frequency of events versus virus concentration.
  • the measured diameter of PBCV-1 (203 ⁇ 14 nm) had a standard deviation (STD) of ⁇ 7%.
  • STD standard deviation
  • Previous reports in the literature on using resistive-pulse sensing to size virus particles have resulted in STDs of ⁇ 4 %.
  • the STD of ⁇ 7% reported here may be due to one of following three effects, or to a combination of these effects.
  • the data used to create the histogram in FIG. 25A was collected from 5 separate experiments that were conducted over seven days. Although the procedure for the experiments was always the same, there may have been small differences (e.g., in temperature or recording buffer) that caused an increase in the STD.
  • FIG. 25 graphically depicts: (A) Histogram of the peak amplitudes of 1395 events caused by PBCV-1 without antibody bound passing through the pore shown in FIGS. 19A, B. The histogram was fit with a Gaussian distribution. (B) Frequency of events versus the concentration of virus. The data points were fit using a linear regression algorithm that required the line to pass through the origin; the slope of the line was 4.0x10 '9 HzxmLxvirus particles "1 .
  • the original serum contained at least 0.55 mgxmL '1 of specific and active antibody. This lower bound compares favorably to a previous study that reported an average concentration of specific antibody of 0.78 mgxmL '1 in rabbit antiserum.
  • FIG. 26 shows microscopic observation of antiserum, control serum, and of virus antibody complexes.
  • TEM Transmission electron microcopy
  • the average distance between viruses in the aggregate was 23 ⁇ 7 nm which is close to the maximum span ( ⁇ 15 nm) of an IgG molecule (see FIG. 27).
  • the serum was used at a dilution of 0.001 and PBCV-1 was used at a concentration of 1x10 9 particlesxmL '1 .
  • Scale bar 100 nm.
  • the buffer used for all images was composed of 150 mM KCI, 50 mM tris(hydroxymethyl)aminomethane (TRIS) buffer, pH 7.8.
  • FIG. 27 shows a TEM image with individual measurements of the distance between virus particles in an aggregate.

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Abstract

La présente invention concerne des procédés et compositions pour détecter l'ensemble de complexes qui consistent à fournir une solution où une première partie est séparée d'une seconde partie par l'intermédiaire d'un pore submicrométrique, d'un tube ou canal submicrométrique, d'un nanopore ou d'un nanotube ou d'un nanocanal. Un ou plusieurs objets submicrométriques ou nanométriques sont ajoutés à la première partie de la solution. En raison des interactions moléculaires, ces objets s'assemblent pour former des complexes se composant de deux ou plusieurs objets submicrométriques ou nanométriques. Le passage d'un complexe de la première partie de la solution à travers le pore submicrométrique, le tube ou canal submicrométrique, le nanopore ou le nanotube ou le nanocanal à la seconde partie de la solution est détecté en utilisant un détecteur impulsionnel à effet résistif. Cette méthodologie de détection peut comprendre la détection de la formation de complexes en temps réel et/ou peut comprendre la détection des complexes pré-assemblés.
PCT/US2007/003133 2006-02-06 2007-02-06 Utilisation de detecteur impulsionnel a effet resistif avec des pores submicrometriques ou nanometriques pour la detection d'ensemble d'objets submicrometriques ou nanometriques WO2007092434A2 (fr)

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US76575806P 2006-02-06 2006-02-06
US60/765,758 2006-02-06
US11/671,171 US20070202495A1 (en) 2006-02-06 2007-02-05 Use of resistive-pulse sensing with submicrometer pores or nanopores for the detection of the assembly of submicrometer or nanometer sized objects
US11/671,171 2007-02-05

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WO2007092434A8 (fr) 2008-01-10
WO2007092434A3 (fr) 2007-11-22

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